Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus

Collart, F.; Osipiuk, J.; Trent, Jonathan D.; Olsen, Gary J.; Huberman, Eliezer

doi:10.1016/0378-1119(96)00044-3

Cited by 9 publications

(5 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…IMPDH shows similarity to GMPR and it has been suggested that both genes are the product of an early duplication of the IMPDH locus [372,373]. Our analyses revealed and verified previous analyses [359] that IMPDH E s of trypanosomatids group within the core group of eukaryotic IMPDHs.…”

Section: Impdh Genessupporting

confidence: 87%

The early evolution of meiotic genes

Malik¹

View full text Add to dashboard Cite

Thesis Supervisor: Associate Professor John M. Logsdon Jr.Meiosis is a hallmark of eukaryotes. However, to demonstrate that homologous sexual processes are pan-eukaryotic, it is necessary to compare the molecular machinery of meiotic recombination across diverse eukaryotic groups, especially protists. The homologs of 34 meiotic genes encoding components of the meiotic recombination machinery, sister chromatid cohesion and synaptonemal complexes were identified in the genomes of diverse eukaryotes and verified by phylogenetic analyses. Twelve of these genes function only in meiosis in model organisms Hop1, Hop2, Mnd1, Dmc1, Msh4, Msh5, Mer3, Zip1, Zip4 and Rec8). Homologs of meiosis-specific genes were identified in several organisms previously considered to be asexual, indicating that they are derived from sexual lineages, and may have as-yet-unobserved meiosis. The putatively early-diverged protists Trichomonas and Giardia have orthologs of eight and six of the meiosis-specific genes, respectively. Using degenerate PCR, additional meiosis-specific genes were identified in Naegleria, Acanthamoeba, Amphidinium and other protists. All of the meiotic genes are conserved in animals, fungi and plants; most of the meiotic genes (including the meiosis-specific genes) are also broadly conserved among protist lineages. These data indicate that the molecular machinery for meiotic recombination evolved once, early during eukaryotic evolution, prior to the divergence of major eukaryotic lineages. Thus, the eukaryotic cenancestor likely had each component of this "meiosis detection toolkit". Many of the 34 meiotic genes -including ten meiosis-specific genes -evolved by gene duplications early during eukaryotic evolution, revealing that gene duplication has been a pervasive evolutionary force in the evolution of the meiotic machinery. Abstract Approved: ____________________________________ Thesis Supervisor ____________________________________ ii To my grandmothers, Bano-bai Mazher Master binte Imran-bhai Shamsi and the late Zainab-bai Gulamali Malik binte Noman-bhai Rangwala, and to my mother, Nafisa Hatim Malik binte Mazher-bhai Master. iii ACKNOWLEDGMENTS I would like to express my gratitude towards the many people who contributed towards my growth as a scientist while I have pursued this degree, from Emory University to the University of Iowa. First and foremost, I would like to thank my thesis advisor, John Logsdon, for his time, advice, many discussions, and for giving me the opportunity to work with him on the projects culminating in this thesis. I also feel fortunate that John has sent me to present my work at a conference almost every year, and really helped me to network with other scientists. I am grateful to my other thesis advisory committee members, Bryant McAllister, Bob Malone, Debashish Bhattacharya and Chris Brochu, for their time, constructive criticism and helpful advice. I am indebted to Mark Farmer and David Patterson for illustrative discussions about protists. I am thankful to Chris Beck, Sonia Altiz...

show abstract

Section: Impdh Genessupporting

confidence: 87%

The early evolution of meiotic genes

Malik¹

View full text Add to dashboard Cite

show abstract

“…A deep branching of the bacterial and eukaryotic forms of IMPDH is observed upon phylogenetic analysis of the relationships among the various IMPDH genes. 13,14 The analysis indicates a general functional conservation of amino acid residues and suggests unique amino acid sequence signature for these kingdoms. The phylogenetic differences between IMPDH enzymes reflect their kinetic differences and differential sensitivity to inhibitors.…”

Section: Introductionmentioning

confidence: 93%

“…aureus ATCC12600 was grown in BHI at 37 ºC up to late log phase [optical density at 540 nm (OD 540 =0.9)]. From the culture the cytosolic fraction was isolated 13 and used for enzyme kinetics of IMPDH. The 3 mL reaction mixture was prepared using 50 mM Tris HCl buffer (pH 8), 1 mM KCl, 1 mM dithiothreitol, 1 mM Ethylene diamine tetraacetic acid (EDTA), 0.05 mM NAD, 10 µM IMP and 50 µl of cytosolic fraction and incubated for 60 minutes at 37 ºC.…”

Section: Enzyme Kinetics Of Impdhmentioning

confidence: 99%

Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation

Yeswanth¹,

Kumar²,

Swarupa³

et al. 2013

JCSR

View full text Add to dashboard Cite

Background: In Staphylococcus aureus purine metabolism plays a crucial role in the formation of biofilm which is a key pathogenic factor. The present study is aimed in the characterization of inosine monophosphate dehydrogenase (IMPDH) from Staphylococcus aureus ATCC 12600.Methods: IMPDH gene was amplified using primers designed from IMPDH gene sequence of S. aureus reported in the database. Then polymerase chain reaction (PCR) product was cloned in the Sma I site of M13mp18 and expressed in Escherichia coli JM109. The recombinant IMPDH (rIMPDH) was overexpressed with 1 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG); Michaelis constant (Km), maximum enzyme velocity (Vmax) and catalytic constant (Kcat) of expressed IMPDH were determined. Results:The enzyme kinetics of IMPDH grown under aerobic conditions showed a Km of 43.71±1.56 µM, Vmax of 0.247±0.84/µM/mg/min and Kcat of 2.74±0.015/min while in anaerobic conditions the kinetics showed Km of 42.81±3.154/ µM, Vmax of 0.378±0.036 µM/mg/min and Kcat of 4.78±0.021/min, indicating elevated levels of IMPDH activity under anaerobic conditions. Three-folds increased activity in the presence of 1 mM adenosine triphosphate (ATP) correlated with biofilm formation. The kinetics of pure rIMPDH were close to the native IMPDH of S. aureus ATCC12600 and the enzyme showed single band in sodium dodecyl sulphate polyacrylamide gel electrophoresis with a molecular weight of 53 KDa.Conclusions: Elevated activity of IMPDH was observed in S. aureus grown under anaerobic conditions and this was correlated with the biofilm formation indicating the linkage between purine metabolism and pathogenesis.

show abstract

“…IMPDH catalyzes the oxidation of IMP to XMP (11-13; reviewed in Reference 14), which is the rate-limiting step in the de novo guanine nucleotide biosynthesis pathway and, as a result, is essential for the growth and proliferation of every cell. Although IMPDH is evolutionarily conserved (15,16), the bacterial enzyme is orders of magnitude more resistant than mammalian IMPDH to the toxic effect of MPA (17)(18)(19)(20). Based on this property, we have demonstrated that expression of bacterial IMPDH in mammalian cells confers in them resistance to MPA toxicity and, moreover, that this enzyme is a useful dominant selectable marker for the expression and selection of exogenous genes or cDNAs in mammalian cell transfections.…”

Section: Introductionmentioning

confidence: 94%

Bacterial IMPDH Gene Used for the Selection of Mammalian Cell Transfectants

Baccam

Huberman

2003

BioTechniques

Self Cite

View full text Add to dashboard Cite

Stable cell transfection is used for the expression of exogenous genes or cDNAs in eukaryotic cells. Selection of these transfectants requires a dominant selectable marker. A variety of such markers has been identified and is currently in use. However, many of these are not suitable for all cell types or require unique conditions. Here we describe a simple and versatile dominant selectable marker that involves bacterial IMP dehydrogenase (IMPDH), an enzyme essential for the replication of mammalian and bacterial cells. Although IMPDH is evolutionarily conserved, the bacterial enzyme is orders of magnitude more resistant to the toxic effect of the drug mycophenolic acid, which is an IMPDH inhibitor. We have demonstrated that transfection of human, monkey or Chinese hamster cell lines with an expression vector containing bacterial IMPDH and mycophenolic acid treatment resulted in the selection of colonies with a strikingly increased resistance to mycophenolic acid toxicity. Analysis of cells derived from these colonies indicated that the acquisition of this resistance was associated with bacterial IMPDH protein expression. As a proof of principle, we showed that mammalian cell transfection with a bicistronic IMPDH/GFP expression vector and mycophenolic acid treatment can be used to successfully select transfectants that express the fluorescent protein. These results indicate that bacterial IMPDH is a practical dominant selectable marker that can be used for the selection of transfectants that express exogenous genes or cDNAs in mammalian cells.

show abstract

Cloning, characterization and sequence comparison of the gene coding for IMP dehydrogenase from Pyrococcus furiosus

Cited by 9 publications

References 25 publications

The early evolution of meiotic genes

The early evolution of meiotic genes

Characterization of inosine monophosphate dehydrogenase from Staphylococcus aureus ATCC12600 and its involvement in biofilm formation

Bacterial IMPDH Gene Used for the Selection of Mammalian Cell Transfectants

Contact Info

Product

Resources

About